| How to refold proteins into correct conformation with high efficiency is one of the industrial bottlenecks in production of recombinant proteins expressed as inclusion bodies (IBs), and formation of correct disulfide bonds is a critical step for the refolding of proteins with many pairs of disulfide bonds. In this thesis, thiol-carrying latex particles were synthesized and then applied to assis the refolding of lysozyme and y-isoenzyme of pig liver esterase (y-PLE). The sulfhydryl groups on microsphere can attack disulfide bonds in misfolded protein to form correct pairing through thiol/disulfide exchange reactions.Firstly, poly(styrene-co-glycidyl methacrylate) particles were synthesized by a two-stage soap-free emulsion polymerization and then reacted with DTT to obtain three kinds of thiol-carrying latex particles (SG-DTT) with different concentrations of sulphydryl groups (4.3μmol/g,15.1μmol/g and79.7μmol/g respectively). Lysozyme was employed as a model protein for refolding process. The results indicated that thiol-carrying latex particles could indeed assist lysozyme refolding and the activity recovery increased from58%to84%with the assistance of SG-DTT particles when initial lysozyme concentration was1000μg/mL. The particles with more sulphydryl groups were more effective when GSSG concentration was0.5mM, and the optimal refolding condition of lysozyme was the concentration ratio of particles to lysozyme of0.5, refolding temperature of15℃and urea concentration of2.5M.Secondly, the γ-PLE was produced by cultivation of the genetic engineering E. coli, and the yield of the γ-PLE IBs was approximately2.0g/L culture. After washed and denatured, the purity of γ-PLE was up to80%. Dilution refolding of γ-PLE was carried out and the effect of concentration of GSSG and GSH was studied. The appropriate concentration of GSSG for γ-PLE refolding increased with protein concentration, and the optimal ratio of GSH to GSSG was in the range of2～4, which was lower than that for lysozyme refolding since lysozyme contained4pairs of disulfide bonds.Finally, based upon simple dilution refolding, three kinds of thiol-carrying latex particles (SG-DTT) with different concentrations of sulphydryl groups (6.1μmol/g,25.2μmol/g and143.4μmol/g respectively) were applied to assist the refolding of y-PLE IBs in vitro. It was shown that particles with higher concentration of sulphydryl groups were more effective and the final y-PLE activity could be1187U/L and1885U/L when y-PLE concentration was500μg/mL and1000μg/mL, which increased by81%and72%respectively than that in refolding with the absence of SG-DTT particles. The optimal refolding condition was the particle concentration of0.5mg/mL, pH of9.5, temperature of15℃and urea concentration of1M.All results above demonstrated that thiol-carrying latex particles could highly increase the refolding efficiency. With the advantages of low cost, easy handling and recycling, thiol-carrying latex particles present potential application in refolding of recombinant proteins which contain disulfide bonds. |
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